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Small-molecule inhibitors of hypoxia-inducible factor prolyl hydroxylases (HIF-PHs) are currently under clinical development as novel treatment options for chronic kidney disease (CKD) associated anemia. Inhibition of HIF-PH mimics hypoxia and leads to increased erythropoietin (EPO) expression and subsequently increased erythropoiesis. Herein we describe the discovery, synthesis, structure-activity relationship (SAR), and proposed binding mode of novel 2,4-diheteroaryl-1,2-dihydro-3H-pyrazol-3-ones as orally bioavailable HIF-PH inhibitors for the treatment of anemia. High-throughput screening of our corporate compound library identified BAY-908 as a promising hit. The lead optimization program then resulted in the identification of molidustat (BAY 85-3934), a novel small-molecule oral HIF-PH inhibitor. Molidustat is currently being investigated in clinical phase III trials as molidustat sodium for the treatment of anemia in patients with CKD.

Plasma levels of FGF23 are increased in patients with chronic kidney disease. Beside its role in phosphate homeostasis, iron deficiency and anemia are associated with increased FGF23 plasma levels. Recently, FGF23 plasma levels were shown to be increased in mice after treatment with hypoxia inducible factor-proline hydroxylase (HIF-PH) inhibitors which are strong inducers of erythropoietin and erythropoiesis and are known to modulate iron uptake and availability. Therefore we investigated a potential context between expression of FGF23 and stimulation of erythropoiesis using a HIF-PH inhibitor and erythropoietin in rats. FGF23 plasma levels are induced at peak levels 2 h after intravenous injection of recombinant human Erythropoietin (rhEPO). Likewise induction of endogenous EPO using a HIF-PH inhibitor (BAY 85-3934) is followed by an increase of FGF23 plasma levels. In contrast to rhEPO the HIF-PH inhibitor induces lower peak levels of FGF23 applying equivalent hematopoietic doses. Bone and bone marrow were identified as sources of EPO-induced FGF23. Immediate induction of FGF23 mRNA was also detected in EPO receptor positive murine hematopoietic BAF3 cells after treatment with rhEPO but not after treatment with the HIF-PH inhibitor. Pretreatment of mice with a neutralizing anti-EPO antibody abrogated FGF23 induction by the HIF-PH inhibitor. Thus, direct impact on FGF23 expression by HIF-PH inhibition in vivo via hypoxia mimicking and modulation of iron metabolism appears unlikely. Collectively, the findings point to an EPO dependent regulation pathway of FGF23 gene expression which might be important in the context of erythropoiesis stimulating therapies in patients with renal anemia.

Cellular oxygen partial pressure is sensed by a family of prolyl-4-hydroxylase domain (PHD) enzymes that modify hypoxia-inducible factor (HIF)α subunits. Upon hydroxylation under normoxic conditions, HIFα is bound by the von Hippel-Lindau tumor suppressor protein and targeted for proteasomal destruction. Since PHD activity is dependent on oxygen and ferrous iron, HIF-1 mediates not only oxygen- but also iron-regulated transcriptional gene expression. Here we show that copper (CuCl2) stabilizes nuclear HIF-1α under normoxic conditions, resulting in hypoxia-response element (HRE)-dependent reporter gene expression. In in vitro hydroxylation assays CuCl2 inhibited prolyl-4-hydroxylation independently of the iron concentration. Ceruloplasmin, the main copper transport protein in the plasma and a known HIF-1 target in vitro, was also induced in vivo in the liver of hypoxic mice. Both hypoxia and CuCl2 increased ceruloplasmin (as well as vascular endothelial growth factor [VEGF] and glucose transporter 1 [Glut-1]) mRNA levels in hepatoma cells, which was due to transcriptional induction of the ceruloplasmin gene (CP) promoter. In conclusion, our data suggest that PHD/HIF/HRE-dependent gene regulation can serve as a sensory system not only for oxygen and iron but also for copper metabolism, regulating the oxygen-, iron- and copper-binding transport proteins hemoglobin, transferrin, and ceruloplasmin, respectively. (Blood. 2005;105:4613-4619)

Interactions between Ets family members and a variety of other transcription factors serve important functions during development and differentiation processes, e.g. in the hematopoietic system. Here we show that the endothelial basic helix-loop-helix PAS domain transcription factor, hypoxia-inducible factor-2alpha (HIF-2alpha) (but not its close relative HIF-1alpha), cooperates with Ets-1 in activating transcription of the vascular endothelial growth factor receptor-2 (VEGF-2) gene (Flk-1). The receptor tyrosine kinase Flk-1 is indispensable for angiogenesis, and its expression is closely regulated during development. Consistent with the hypothesis that HIF-2alpha controls the expression of Flk-1 in vivo, we show here that HIF-2alpha and Flk-1 are co-regulated in postnatal mouse brain capillaries. A tandem HIF-2alpha/Ets binding site was identified within the Flk-1 promoter that acted as a strong enhancer element. Based on the analysis of transgenic mouse embryos, these motifs are essential for endothelial cell-specific reporter gene expression. A single HIF-2alpha/Ets element conferred strong cooperative induction by HIF-2alpha and Ets-1 when fused to a heterologous promoter and was most active in endothelial cells. The physical interaction of HIF-2alpha with Ets-1 was demonstrated and localized to the HIF-2alpha carboxyl terminus and the autoinhibitory exon VII domain of Ets-1, respectively. The deletion of the DNA binding and carboxyl-terminal transactivation domains of HIF-2alpha, respectively, created dominant negative mutants that suppressed transactivation by the wild type protein and failed to synergize with Ets-1. These results suggest that the interaction between HIF-2alpha and endothelial Ets factors is required for the full transcriptional activation of Flk-1 in endothelial cells and may therefore represent a future target for the manipulation of angiogenesis.

Oxygen sensing by hypoxia-inducible factor prolyl hydroxylases (HIF-PHs) is the dominant regulatory mechanism of erythropoietin (EPO) expression. In chronic kidney disease (CKD), impaired EPO expression causes anemia, which can be treated by supplementation with recombinant human EPO (rhEPO). However, treatment can result in rhEPO levels greatly exceeding the normal physiological range for endogenous EPO, and there is evidence that this contributes to hypertension in patients with CKD. Mimicking hypoxia by inhibiting HIF-PHs, thereby stabilizing HIF, is a novel treatment concept for restoring endogenous EPO production. HIF stabilization by oral administration of the HIF-PH inhibitor BAY 85-3934 (molidustat) resulted in dose-dependent production of EPO in healthy Wistar rats and cynomolgus monkeys. In repeat oral dosing of BAY 85-3934, hemoglobin levels were increased compared with animals that received vehicle, while endogenous EPO remained within the normal physiological range. BAY 85-3934 therapy was also effective in the treatment of renal anemia in rats with impaired kidney function and, unlike treatment with rhEPO, resulted in normalization of hypertensive blood pressure in a rat model of CKD. Notably, unlike treatment with the antihypertensive enalapril, the blood pressure normalization was achieved without a compensatory activation of the renin–angiotensin system. Thus, BAY 85-3934 may provide an approach to the treatment of anemia in patients with CKD, without the increased risk of adverse cardiovascular effects seen for patients treated with rhEPO. Clinical studies are ongoing to investigate the effects of BAY 85-3934 therapy in patients with renal anemia.

Selective inhibitors of Jumonji domain-containing protein (JMJD) histone demethylases are candidate anticancer agents as well as potential tools for elucidating the biological functions of JMJDs. On the basis of the crystal structure of JMJD2A and a homology model of JMJD2C, we designed and prepared a series of hydroxamate analogues bearing a tertiary amine. Enzyme assays using JMJD2C, JMJD2A, and prolyl hydroxylases revealed that hydroxamate analogue 8 is a potent and selective JMJD2 inhibitor, showing 500-fold greater JMJD2C-inhibitory activity and more than 9100-fold greater JMJD2C-selectivity compared with the lead compound N-oxalylglycine 2. Compounds 17 and 18, prodrugs of compound 8, each showed synergistic growth inhibition of cancer cells in combination with an inhibitor of lysine-specific demethylase 1 (LSD1). These findings suggest that combination treatment with JMJD2 inhibitors and LSD1 inhibitors may represent a novel strategy for anticancer chemotherapy.

The abundance of the transcription factor hypoxia-inducible factor is regulated through hydroxylation of its alpha-subunits by a family of prolyl-hydroxylases (PHD1-3). Enzymatic activity of these PHDs is O2-dependent, which enables PHDs to act as cellular O2 sensor enzymes. Herein we studied endogenous PHD activity that was induced in cells grown under hypoxia or in the presence of nitric oxide. Under such conditions nuclear extracts contained much higher PHD activity than the respective cytoplasmic extracts. Although PHD1-3 were abundant in both compartments, knockdown experiments for each isoenzyme revealed that nuclear PHD activity was only due to PHD2. Maximal PHD2 activity was found between 120 and 210 microm O2. PHD2 activity was strongly decreased below 100 microm O2 with a half-maximum activity at 53 +/- 13 microm O2 for the cytosolic and 54 +/- 10 microm O2 for nuclear PHD2 matching the physiological O2 concentration within most cells. Our data suggest a role for PHD2 as a decisive oxygen sensor of the hypoxia-inducible factor degradation pathway within the cell nucleus.

N-nitrosomelatonin (NOMela) is well-known for its capabilities of transnitrosating nucleophiles such as thiols and ascorbate, thereby generating nitric oxide (NO)-releasing compounds. It is unknown, however, whether NOMela can be successfully applied as a precursor of NO in a complex biological environment like a cell culture system. NO donors may be useful to induce the transcription factor hypoxia inducible factor 1 (HIF-1), which coordinates the protection of cells and tissues from the lack of oxygen (hypoxia). In this study, the effects of NOMela in an in vitro cell-free assay [NO-release, inhibition of prolylhydroxylase1 (PHD1)] and in living cells (upregulation of HIF-1, reduction of HIF-1 hydroxylation, upregulation of the HIF-1-target gene PHD2) were compared with those of the frequently applied NO donor S-nitrosoglutathione (GSNO) under normoxic and hypoxic conditions. In contrast to GSNO, NOMela released NO in a predictable manner and this release in vitro was found to be independent of the composition of the buffer system. The NOMela-mediated effects in oxygenated cells were in all cases comparable to the hypoxic response, whereas unphysiological strong effects were observed with GSNO. Probably, because of the antioxidative power of the NOMela-dependent formation of melatonin, cells were completely protected against the attack of reactive nitrogen oxygen species, which are generated by autoxidation of NO. In conclusion, NOMela had to be an excellent NO precursor for cells in culture and potentially tissues.

The prolyl-4-hydroxylase domain (PHD) oxygen sensor proteins hydroxylate hypoxia-inducible transcription factor (HIF)-alpha (alpha) subunits, leading to their subsequent ubiquitinylation and degradation. Since oxygen is a necessary cosubstrate, a reduction in oxygen availability (hypoxia) decreases PHD activity and, subsequently, HIF-alpha hydroxylation. Non-hydroxylated HIF-alpha cannot be bound by the ubiquitin ligase von Hippel-Lindau tumor suppressor protein (pVHL), and HIF-alpha proteins thus become stabilized. HIF-alpha then heterodimerizes with HIF-beta (beta) to form the functionally active HIF transcription factor complex, which targets approximately 200 genes involved in adaptation to hypoxia. The three HIF-alpha PHDs are of a different nature compared with the prototype collagen prolyl-4-hydroxylase, which hydroxylates a mass protein rather than a rare transcription factor. Thus, novel assays had to be developed to express and purify functionally active PHDs and to measure PHD activity in vitro. A need also exists for such assays to functionally distinguish the three different PHDs in terms of substrate specificity and drug function. We provide a detailed description of the expression and purification of the PHDs as well as of an HIF-alpha-dependent and a HIF-alpha-independent PHD assay.

Hypoxia-inducible factors (HIFs) are transcriptional regulators that mediate the cellular response to low oxygen levels. By stimulating the expression of angiogenic growth factors such as vascular endothelial growth factor (VEGF), they trigger the neovascularization of tissues under physiologic and pathologic conditions. Here, we have investigated the endothelial cell–autonomous HIF function in blood vessel growth and development by expressing a dominant-negative HIF mutant (HIFdn) that inhibits the transcriptional responses mediated by both HIF-1 and HIF-2, specifically in endothelial cells of transgenic mice. HIFdn transgenic embryos were growth retarded and died around E11.5. Primitive vascular networks were established, but vascular remodeling in the yolk sac and in the embryo proper was defective, and vascular sprouts failed to invade the neuroepithelium. In addition, heart looping was incomplete, and the ventricles of the heart were thin-walled and lacked trabeculation. Similar cardiovascular defects have been observed in Tie2–deficient mouse embryos. Consistently, HIFdn transgenic embryos expressed reduced levels of the endothelial angiopoietin receptor, Tie-2, whereas other endothelial markers, such as PECAM-1, Tie-1, and VE-cadherin were not affected. These results show that HIFs in endothelial cells are essential for embryonic heart and blood vessel development and control angiogenesis and vascular remodeling.

The transcriptional activation of hypoxia-inducible genes is essential for the adaptation of mammalian tissues to oxygen deficiency. The hypoxia-inducible transcription factor (HIF) is a cellular switch for the up-regulation of these genes during hypoxia. Under normoxia, HIFs are hydroxylated on conserved prolyl residues by a recently discovered family of HIF prolyl hydroxylases (HIF-PHD1-3). Hydroxylated HIF specifically interacts with the von Hippel–Lindau protein–elongin B–elongin C complex (VBC) which leads to ubiquitination and subsequent proteasomal degradation of HIF. We developed a nonradioactive microtiter plate assay based on the interaction of hydroxylated HIF with VBC which enabled us to detect hydroxylated HIF in the nanomolar concentration range. A biotinylated HIF peptide substrate was bound to a streptavidin-coated microtiter plate and hydroxylated with the HIF-PHD3 isoenzyme. Recombinant VBC complex with a thioredoxin (Trx) tag was purified from Escherichia coli and bound to the hydroxylated HIF peptide. The interaction between VBC and hydroxylated HIF was detected by using an anti-thioredoxin antibody.

Hypoxia-inducible transcription factors (HIFs) are important for transcriptional adaptation to hypoxia. Availability of HIFs is regulated via posttranslational modification of their alpha subunits (HIF-1alpha and HIF-2alpha). Under normoxia, two highly conserved proline residues within the oxygen-dependent degradation domain (ODDD) are hydroxylated by oxoglutarate-dependent proline 4-hydroxylases EGLN1-3. Hydroxylated HIF-alpha interacts with the pVHL-E3 ubiquitin ligase complex and, subsequently, is degraded via the proteasomal pathway. We identified a novel putative proline 4-hydroxylase, PH-4, with an aminoterminal EF-hand motif and a carboxyterminal catalytic domain, which was highly expressed in most organs, and-unlike EGLNs which localize to the cytoplasm and nucleus-was associated with the endoplasmic reticulum. Like EGLNs, PH-4 overexpressed in cellular reporter assays suppressed the HIF transactivation activity, dependent on the consensus ODDD proline residues. Suppression of transactivation was correlated with decrease of cellular contents of HIF. Thus, PH-4 might be related to cellular oxygen sensing.

Degradation of angiogenic mediators might be an underlying cause of chronic wounds. To test this hypothesis, we evaluated the expression and integrity of vascular endothelial growth factor, a potent angiogenic mediator, and its receptors, Flt-1 and KDR, in chronic venous leg ulcerations. Immunohisto- chemical, in situ hybridization, and semiquantitative reverse transcriptase polymerase chain reaction analyses all indicate that expression of vascular endothelial growth factor is elevated in ulcerative tissue, with vascular endothelial growth factor mRNA being especially pronounced in the hyperplastic epithelium of the wound margin. Flt-1 and KDR protein and mRNA were detected in the papillary vessels in close vicinity to the lesional epithelium of chronic wounds. Although increased expression of vascular endothelial growth factor protein was detected in the epidermis, the intensity of this staining was weak compared with the epidermal staining in psoriatic lesions and compared with the strong vascular endothelial growth factor mRNA signal in chronic wounds and psoriasis. To analyze whether this apparent decrease in immunoreactivity could be the result of degradation of vascular endothelial growth factor by proteolytic activities from the wound environment, we examined the stability of recombinant vascular endothelial growth factor in wound fluid from chronic leg ulcers. As demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, incubation of rVEGF165 with chronic, but not acute, wound fluid resulted in rapid proteolytic degradation of rVEGF165. Protease inhibitor studies indicate that serine proteases, such as plasmin, are involved in this degradation. Together, our data show that, although vascular endothelial growth factor expression is elevated in chronic wounds, increased proteolytic activity in this environment results in its degradation, which may contribute to an impaired wound healing response.

The vascular endothelial growth factor (VEGF) receptor-2 (Flk-1) is the first endothelial receptor tyrosine kinase to be expressed in angioblast precursors, and its function is essential for the differentiation of endothelial cells and hematopoietic precursors. We have identified cis-acting regulatory elements of the murineFlk-1 gene that mediate endothelium-specific expression of a LacZ reporter gene in transgenic mice. Sequences within the 5′-flanking region of the Flk-1 gene, in combination with sequences located in the first intron, specifically targeted transgene expression to angioblasts and endothelial cells of transgenic mice. The intronic regulatory sequences functioned as an autonomous endothelium-specific enhancer. Sequences of the 5′-flanking region contributed to a strong, uniform, and reproducible transgene expression and were stimulated by the transcription factor HIF-2. The Flk-1 gene regulatory elements described in this study should allow the elucidation of the molecular mechanisms involved in endothelial cell differentiation and angiogenesis.